Polyketides represent a singularly useful group of natural products which are related by their general pathway of biosynthesis. Representative members include the macrolide antibiotics, for example, erythromycin, spiramycin and tylosin, immunosuppressants such as rapamycin and FK506, antiparasitics such as the avermectins, antifungal agents such as amphotericin B and nystatin, anticancer agents such as daunorubicin and doxorubicin and anticholesterolemics such as mevinolin. Polyketides generally are secondary metabolites of the actinomycetes including the genera Streptomyces, Actinomyces, Actinomadura, Micromonospora, Saccharopolyspora, and Nocardia. It was estimated that in 1986 about 6,000 antibiotics of microbial origin had been characterized of which 70 were in clinical use; an additional 1100 metabolites were reported between 1988 and 1992, approximately 40% of which were polyketides.
Despite the multiplicity of polyketide structures available from nature, there remains a need to expand the repertoire of available polyketides and to synthesize a multiplicity of polyketides in the form of libraries so that there is a convenient substrate for screening to identify polyketides that are relevant to a specific target of interest. The present invention provides solutions to these needs.
Polyketides generally are synthesized by condensation of two-carbon units in a manner analogous to fatty acid synthesis. In general, the synthesis involves a starter unit and extender units; these “two-carbon” units are derived from acylthioesters, typically acetyl, propionyl, malonyl or methylmalonyl coenzyme-A thioesters. There are two major classes of polyketide synthases (PKSs) which differ in the “manner” in which the catalytic sites are used—the so-called “aromatic” PKS and the modular PKS. The present invention employs coding sequences from both these classes as will further be explained below.
Recombinant production of heterologous functional PKS—i.e., a PKS which is capable of producing a polyketide—has been achieved in Streptomyces and hybrid forms of aromatic PKSs have been produced in these hosts as well. See, for example, Khosla, C. et al. J Bacteriol (1993) 175:2194–2204; Hopwood, D. A. et al. Nature (1985) 314:642–644; Sherman, D. H. et at. J Bacteriol (1992) 174:6184–6190. In addition, recombinant production of modular PKS enzymes has been achieved in Streptomyces as described in PCT application WO 95/08548. In all of these cases, the PKS enzymes have been expressed from a single vector. A single vector which carried genes encoding PKS catalytic sites was transformed into E. coli by Roberts, G. A., et al., Eur J Biochem (1993) 214:305–311, but the PKS was not functional, presumably due to lack of pantetheinylation of the acyl carrier proteins.
The present invention provides double or multivector systems for production of PKS and the resultant polyketides in a variety of hosts. The use of multiple vectors provides a means more efficiently to enhance the number of combinatorial forms of PKS and polyketides that can be prepared. Addition of the machinery for pantetheinylation of the acyl carrier proteins (i.e., a holo ACP synthase) permits production of polyketides in a wide spectrum of hosts.